JP2021527434A - Polyunsaturated fatty acid composition enriched with DHA - Google Patents

Abstract

A vegetable-based lipid composition comprising high levels of at least three different long-chain polyunsaturated fatty acids (typically as fatty acid esters) is provided. The composition contains DHA as the major long-chain polyunsaturated fatty acid. The composition is available from a single source by conventional processing methods and has improved stability properties.

Description

The embodiments disclosed herein relate to novel lipid compositions enriched with multiple polyunsaturated fatty acids. Polyunsaturated fatty acid compositions have many health benefits, including nutritional benefits, and are potentially derived from a single source that is both expandable and durable. It also has improved stability against oxidation.

Omega-3 long-chain polyunsaturated fatty acids (LC-PUFAs) are widely recognized as important compounds for human and animal health. These fatty acids can be obtained from dietary sources or, to a lesser extent, by the conversion of linoleic acid (LA, 18: 2ω-6) or α-linolenic acid (ALA, 18: 3ω-3) fatty acids. They are sexual and are all considered essential fatty acids in the human diet.

From a nutritional point of view, the most important omega-3 fatty acids are probably α-linolenic acid, eicosapentaenoic acid (“EPA”, 20: 5n-3), and docosahexaenoic acid (“DHA”, 22: 6n-3). Is. DHA is LC-PUFA and is important for brain and eye development. Ingestion of omega-3 PUFA may also help prevent coronary artery disease. Medical studies have clearly shown that these fatty acids have beneficial health aspects such as improved cardiovascular and immune function, or reduced cancer, diabetes, and hypertension. Clinical results demonstrated that a weekly dietary intake of 5.5 g of omega-3 PUFA may be associated with a 50% reduction in the risk of primary cardiac arrest. As a result, oils containing omega-3 PUFA are in high demand for pharmaceutical and nutritional purposes.

In general, the oxidative stability of fatty acids decreases significantly as the number of carbon-carbon double bonds, or degree of unsaturation, increases. Unfortunately, ALA, EPA, and DHA are all polyunsaturated fats and tend to oxidize easily. EPA (with 5 carbon-carbon double bonds) is significantly more likely to be oxidized than ALA, and DHA (with 6 carbon-carbon double bonds) is even more likely to be oxidized than EPA. As a result, increasing the omega-3 content tends to shorten the shelf life of many products. These problems are especially exacerbated by oils containing significant amounts of EPA or DHA.

US2015 / 223483 discloses a canola oil-based blend with improved oxidative stability. Stability is achieved by the addition of one or more additives.

US2011 / 0027443 discloses a fat composition containing a specific blend of oleic acid, linoleic acid, alpha-linolenic acid and LC-PUFA with an improved flavor profile. US2004 / 209953 discloses nutritional products containing predominantly monoglycerides and diglycerides in LC-PUFA. US 5,130,061 describes the use of transesterification and distillation processes to extract DHA from crude oil. US 9,040,730 describes the purification of lipid mixtures containing PUFAs to reduce the amount of unwanted sterols in the composition. In any of these cases, fish oil or microbial oil is used as a raw material to obtain a particular blend.

International Patent Application No. WO 2013/185184 discloses a process for producing ethyl esters of polyunsaturated fatty acids.

International Patent Application WO2015 / 089587 and US Patent Application US2015 / 0166928 disclose plant lipid compositions comprising a mixture of omega-3 and omega-6 fatty acids. Genetically modified canolas are described in WO2017 / 218969 and WO2017 / 219006.

International patent applications WO 2016/182452 and WO 2014/105576 disclose compositions containing significant amounts of DHA and EPA.

The list or discussion of documents apparently previously published herein should not necessarily be construed as acknowledging that the document is part of the latest technology or common general knowledge.

According to the first aspect of the present invention, it is a vegetable-based lipid composition.
(I) Docosahexaenoic acid (22: 6n-3) in an amount of about 50% by weight to about 85% by weight of the total fatty acid content of the composition.
(Ii) With a second polyunsaturated fatty acid in an amount of about 10% to about 90% by weight of docosahexaenoic acid,
(Iii) Containing a third polyunsaturated fatty acid in an amount of about 10% to about 70% by weight of the second polyunsaturated fatty acid.
The total amount of all other fatty acids in the composition is up to about 20% by weight of the total fatty acid content of the composition, and docosahexaenoic acid, each of the second and third polyunsaturated fatty acids, is independent. , Fatty acid, fatty acid salts, fatty acid esters, or fatty acid ester salts are provided, and vegetable-based lipid compositions are provided in which the amount of EPA in the composition is less than 10% of the amount of DHA.

The lipid composition is referred to herein as the "composition of the invention".

The present invention presents high levels of first polyunsaturated fatty acids (DHA) along with at least two other polyunsaturated fatty acids, each of which may be in the form of a free fatty acid, salt, ester, or ester salt. The present invention relates to a lipid composition containing. These compositions have been found to be available from sustainable sources such as botanical sources. They were also found to have an improved storage stability profile demonstrated by reduced degradation due to oxidation during storage. Many polyunsaturated fatty acids, especially DHA, are recognized as important compounds for human and animal health. These compositions can be used in feeds, dietary supplements, cosmetics, and other chemical compositions, which can be useful as intermediates and pharmaceutical active ingredients.

Fatty acid levels in the compositions of the present invention can be determined using conventional methods known to those of skill in the art. Such methods include, for example, gas chromatography (GC) combined with a reference standard according to the methods disclosed in the Examples. In certain methods, fatty acids are converted to methyl or ethyl esters prior to GC analysis. Such techniques will be described in the examples. The peak position of the chromatogram can be used to identify each particular fatty acid and the area under each peak can be integrated to determine the amount. As used herein, the percentage of a particular fatty acid in a sample shall be calculated as the area under the curve of the chromatogram of that fatty acid as the percentage of the total area of the fatty acids in the chromatogram, unless stated conversely. Determined by. This essentially corresponds to a weight percent (w / w). The identity of fatty acids can be confirmed by GC-MS.

The term "polyunsaturated fatty acid" or, as an alternative, "PUFA" refers to a fatty acid that contains at least two carbon-carbon double bonds. The terms "long-chain polyunsaturated fatty acid" and "LC-PUFA" refer to fatty acids whose carbon chain contains at least 20 carbon atoms and at least two carbon-carbon double bonds, and thus VLC- Includes PUFA. As used herein, the terms "very long chain polyunsaturated fatty acid" and "VLC-PUFA" refer to at least 22 carbon atoms and at least 3 carbon-carbon doubles in the carbon chain. Refers to fatty acids containing double bonds. Usually, the number of carbon atoms in the carbon chain of a fatty acid refers to an unbranched carbon chain. If the carbon chain is branched, the number of carbon atoms excludes the carbon atoms of the side group.

The long-chain polyunsaturated fatty acid can be an ω3 (“omega-3”) fatty acid, that is, a fatty acid having an unsaturated (carbon-carbon double bond) at the third carbon-carbon bond from the methyl end of the fatty acid. .. Alternatively, they can be ω6 (“omega-6”) fatty acids, i.e. fatty acids that have an unsaturated (carbon-carbon double bond) at the sixth carbon-carbon bond from the methyl end of the fatty acid. The types of ω6 and especially ω3 are particularly relevant in the context of the present invention, while other unsaturated patterns may be present.

The compositions of the present invention contain at least three different polyunsaturated fatty acids. The first polyunsaturated fatty acid (DHA) is the most abundant fatty acid present in the composition (by weight relative to the total fatty acid content of the composition).

References to docosahexaenoic acid and "DHA" in this context are unsaturated to the ω3 form of docosahexaenoic acid, the third carbon-carbon bond from the methyl end of the fatty acid (carbon-carbon double bond). It is a reference to docosahexaenoic acid having. Abbreviations that can be used synonymously include "22: 6n-3" and "22: 6ω-3".

The DHA and the second and third PUFAs can each be provided independently in the form of a fatty acid, a fatty acid salt, a fatty acid ester, or a salt of a fatty acid ester.

As used herein, the term "fatty acid" often refers to a carboxylic acid (or organic acid) that has a long aliphatic tail, either saturated or unsaturated. Typically, fatty acids have carbon-carbon bond chains that are at least 8 carbon atoms long, and more specifically at least 12 carbons long. Most natural fatty acids have an even number of carbon atoms because they contain acetate, which has two carbon atoms in biosynthesis. Fatty acids are in the free state (non-esterified), referred to herein as "free fatty acids," or alkyl esters, parts of triglycerides, parts of diacylglycerides, parts of monoacylglycerides, acyl-CoA (thioester) bonds. Alternatively, it may be in another bound form, or an esterified form such as a mixture thereof. The fatty acid can be esterified as a phospholipid in the form of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol or diphosphatidylglycerol, but is preferably esterified as an alkyl ester, especially as an ethyl ester. do. For the avoidance of doubt, the term "fatty acid" includes free fatty acids, fatty acid esters, and salts of any of these, unless otherwise stated. Unless otherwise stated, a quantitative value associated with a particular fatty acid refers to the amount of that fatty acid present (calculated based on weight), regardless of the form in which it is present (eg, free acid or ester).

Each fatty acid in the composition may also be provided in the form of a fatty acid salt, such as an alkaline salt or an alkaline earth salt. Specific salts that may be mentioned include lithium and calcium salts. Such salts have potential additional medicinal properties or provide improved processability. Similarly, fatty acid esters can be provided in the form of salts of fatty acid esters. Any combination of fatty acids in the form of free fatty acids, salts, esters, or salts of esters can be present in the compositions of the present invention. This means that, as an example, DHA can be present primarily as an ethyl ester, a second PUFA can be present primarily as a calcium salt of the methyl ester, and a third PUFA can be present primarily as a free fatty acid. do.

"Saturated fatty acids" do not contain any double bonds or other functional groups along the chain. The term "saturated" refers to hydrogen in that all carbons (except the carboxylic acid [-COOH] group) contain as much hydrogen as possible. In other words, the omega (ω) end is _{bound to three} hydrogen atoms (CH 3 −), and each carbon in the chain is bound to two hydrogen atoms (−CH ₂ −). The term "total fatty acid" includes all forms of fatty acids and is saturated or unsaturated, free acids, esters and / or salts.

The term "about" as used herein to refer to measurable values such as the amount, weight, time, temperature of a compound is 20%, 10%, 5%, 1% of the specified amount. Refers to a variation of 0.5%, or even 0.1%.

The compositions of the invention that may be mentioned include those containing high concentrations of omega-3 fatty acids, many of which are so-called "essential fats" that are considered to be of particular importance to human health. Omega-3 fatty acids have been shown to have beneficial effects on HDL cholesterol levels, support adolescent brain development and contribute to mental health. These fatty acids are generally considered to be precursors of anti-inflammatory eicosanoids. In a particular composition of the invention that may be mentioned, the total amount of omega-3 polyunsaturated fatty acids in the lipid composition is at least about 80% by weight, for example at least about 85% by weight, of the total fatty acid content of the composition. Including those that are.

Omega-6 fatty acids are also considered important for human health. In particular, certain omega-6 fatty acids are "essential fats" needed for health, but the body is unable to synthesize them. However, omega-6 fats have been shown to be precursors of more pro-inflammatory eicosanoids, and overproduction of these eicosanoids can increase inflammation and inflammatory disease. Therefore, it may be desirable to minimize the amount of such fatty acids in the lipid composition. It is generally accepted that the ratio of omega-6 fatty acids to omega-3 fatty acids in the diet should be 4: 1 or less. However, a normal Western diet typically has a high proportion of omega-6 fatty acids. The lipid compositions of the present invention advantageously contain small amounts of omega-6 fatty acids and at the same time contain large amounts of more beneficial omega-3 fatty acids. In one embodiment, the total amount of omega-6 polyunsaturated fatty acids in the composition is at most about 10% by weight of the total fatty acid content of the composition. In another embodiment, the ratio of the total weight of omega-3 polyunsaturated fatty acids to the total weight of omega-6 polyunsaturated fatty acids in the composition is at least about 10: 1. In a further embodiment, the ratio of the total weight of omega-3 polyunsaturated fatty acids to the total weight of omega-6 polyunsaturated fatty acids in the composition is at least about 20: 1.

Lipid compositions containing long-chain polyunsaturated fatty acids are typically obtained from marine sources (eg, fish, crustaceans), algae sources, or plant sources (eg, flax or equium). The starting organic matter is first processed to extract the oil contained therein (commonly referred to as "crude" oil). For example, in the case of plant seeds, the seeds are ground to release oil, which is then separated from the solid by filtration and / or decantation. Crude oils often contain levels of polyunsaturated fatty acids (eg, nutritional products) that are too low to be useful and therefore need to be concentrated. When crude oil is deficient in one or more essential ingredients, it is common to blend crude or concentrated oils together from multiple sources (eg, from fish and algae) to obtain the desired composition. .. Alternatively, the concentration is to process the crude oil while maximizing the level of the desired fatty acid component and remove unwanted components (eg, components that adversely affect the color, odor or stability of the product, or unwanted fatty acids). Can be achieved by.

The compositions of the present invention are advantageously available from a single source. The use of a single source facilitates the efficient and economical processing of crude oil and the production of the lipid compositions of the present invention. The phrase "available from a single source" (or "obtained from a single source") is available from one or more organisms whose lipid composition is in a single taxonomic class. Means that. In certain embodiments, the lipid composition is not derived from multiple organisms across different taxonomic classes. For example, the lipid composition should not be a blend of oils obtained from a combination of fish and algae, or a combination of fish and plants. Alternatively, the lipid compositions of the present invention (or "crude" oils from which the composition can be obtained by concentration techniques such as transesterification, distillation, and chromatography) are a single biological population, eg, a single plant. Available from sources or vegetation sources. For the avoidance of doubt, the phrase "available from a single source" refers to the use of multiple organisms of the same species as a source of lipid compositions or "crude" oils, ie multiple fish, algae. It does not preclude the use of resources, plants, or plant seeds of the same species. The plurality of organisms are preferably all of the same species, or of the same breeding line, or of the same plant species, or of the same resource or batch.

In the compositions of the present invention, DHA is present in an amount of about 50% to about 85% by weight of the total fatty acid content of the composition. In one embodiment, DHA is present in an amount that is at least about 55% by weight (eg, at least about 60% by weight) of the total fatty acid content of the composition. In a further embodiment of the invention, DHA is present in an amount of up to about 80% by weight (eg, up to about 75% by weight) of the total fatty acid content of the composition.

The lipid compositions of the present invention advantageously have high levels of DHA compared to the amount of EPA present. Therefore, in one embodiment, the weight ratio of docosahexaenoic acid to eicosapentaenoic acid in the lipid composition is at least about 10: 1. In a further embodiment, the weight ratio of docosahexaenoic acid to eicosapentaenoic acid in the lipid composition can be at least about 20: 1, for example greater than about 30: 1.

Preferably, the amount of EPA in the composition may be very low. In certain embodiments, the lipid composition contains up to about 3% by weight of the total fatty acid content of the composition, more specifically up to about 2% by weight of EPA.

The second polyunsaturated fatty acid can be any polyunsaturated fatty acid disclosed herein, except that the second and third PUFAs differ and neither is DHA. In one embodiment, the second polyunsaturated fatty acid is a C18-24 omega-3 polyunsaturated fatty acid containing at least 3 (eg, at least 4) unsaturated fatty acids. Specific such PUFAs that may be mentioned include α-linolenic acid (“ALA”, 18: 3n-3), eicosatetraenoic acid (“ETA”, 20: 4n-3), docosapentaenoic acid (“ALA”, 20: 4n-3). "DPA", 22: 5n-3), and stearadonic acid ("SDA", 18: 4n-3) are included. In yet another embodiment, the second polyunsaturated fatty acid is ALA, ETA, or DPA.

The compositions of the present invention contain an amount of about 10% to about 90% by weight of DHA of the second polyunsaturated fatty acid. In one embodiment, the second polyunsaturated fatty acid is the second most abundant polyunsaturated fatty acid in the composition. In another embodiment, the second polyunsaturated fatty acid is present in an amount of about 10% to about 60% by weight of DHA.

The third polyunsaturated fatty acid can be any polyunsaturated fatty acid disclosed herein, except that the second and third PUFAs differ and neither is DHA. In one embodiment, the third polyunsaturated fatty acid is a C18-24 omega-3 polyunsaturated fatty acid that contains at least three (eg, at least four) unsaturated fatty acids. Specific such PUFAs that may be mentioned include ALA, EPA, ETA, DPA, and SDA. In yet another embodiment, the third polyunsaturated fatty acid is ETA, EPA, or DPA.

The composition of the present invention contains a third polyunsaturated fatty acid in an amount of about 10% to about 70% by weight of the second PUFA. In one embodiment, the third polyunsaturated fatty acid is the third most abundant polyunsaturated fatty acid in the composition. In another embodiment, the third polyunsaturated fatty acid is present in an amount of about 10% to about 65% by weight of the second PUFA.

The compositions of the present invention may also contain other polyunsaturated fatty acids (ie, in addition to DHA and the second and third PUFAs). Such additional polyunsaturated fatty acids include those described above for a third PUFA (eg, ALA, EPA, ETA, DPA, and / or SDA), as well as linoleic acid (“LA”, 18). : 2n-6) and y-linolenic acid (“GLA”, 18: 3n-6) are included. In the composition of the present invention, the total amount of all other fatty acids in the composition is up to about 20% by weight of the total fatty acid content of the composition. This means that the total amount of all fatty acids in the composition is about 20% by weight of the total fatty acid content of the composition, free of DHA and the second and third PUFAs. In one particular embodiment, the total amount of all other fatty acids in the composition is up to about 18% by weight (eg, up to about 16% by weight) of the total fatty acid content of the composition.

The composition of the present invention contains palmitic acid (16: 0) up to about 0.5% by weight of the total fatty acid content of the composition. In certain embodiments, the composition contains up to about 0.1% by weight of the total fatty acid content of the composition, more specifically up to about 0.05% by weight of palmitic acid.

Preferences and options for a given aspect, feature, or embodiment of the invention are any and all preferences and options for all other aspects, features, and embodiments of the invention, unless otherwise indicated in the context. It should be considered disclosed in combination with the options. For example, the specific amounts of DHA and second and third PUFAs shown in the previous section are disclosed in all combinations.

Therefore, certain lipid compositions that may be mentioned are:
(I) With DHA in an amount of about 50% by weight to about 85% by weight of the total fatty acid content of the composition.
(Ii) With an amount of ALA of about 10% by weight to about 90% by weight of DHA,
(Iii) Containing a third polyunsaturated fatty acid in an amount of about 10% to about 70% by weight of ALA.
The total amount of all other fatty acids in the composition is up to about 20% by weight of the total fatty acid content of the composition, with DHA and each of the second and third polyunsaturated fatty acids independently. Provided in the form of fatty acids, fatty acid salts, fatty acid esters, or salts of fatty acid esters, the amount of EPA in the composition is less than 10% of the amount of DHA.

Further specific lipid compositions that may be mentioned include
(I) With an amount of DHA of about 55% by weight to about 80% by weight of the total fatty acid content of the composition,
(Ii) With an amount of ALA of about 10% by weight to about 60% by weight of DHA,
(Iii) Containing about 10% to about 70% by weight of ALA, a third polyunsaturated fatty acid selected from ETA and DPA.
The total amount of all other fatty acids in the composition is up to about 20% by weight of the total fatty acid content of the composition, and DHA, ALA, and the third polyunsaturated fatty acid are independent fatty acids. , Fatty acid salts, fatty acid esters, or salts of fatty acid esters, the amount of EPA in the composition is less than 10% of the amount of DHA.

In the compositions of the invention, the DHA and the second and third PUFAs can each independently exist in the form of fatty acids, fatty acid salts, fatty acid esters, or salts of fatty acid esters. In certain embodiments, each of these components takes the same form, for example, they may be in the form of all fatty acids, all in fatty acids, all in fatty acid esters, or all in salts of fatty acid esters. If the component is in the form of a fatty acid salt, ester or ester salt, the component can be in the same salt, ester or ester salt form. For example, DHA and second and third PUFAs can be provided in the form of ethyl esters of fatty acids.

In certain embodiments, the DHA and the second and third PUFAs are each independently provided in the form of a salt of a fatty acid ester, or most specifically in the form of a fatty acid ester. Suitable fatty acid ester forms are known to those of skill in the art. For example, forms of fatty acid esters that are nutritionally acceptable and / or pharmaceutically acceptable include ethyl esters, methyl esters, phospholipids, monoglycerides, diglycerides, and triglycerides of fatty acids. Different ester forms may be required depending on the intended use of the lipid composition. For example, triglycerides are particularly suitable for use in foods intended for human consumption, especially for infant consumption, in part because of these ester forms for heat treatment (which may be required for such foods). It is due to taste and stability. Thus, in one embodiment, a food for human or animal consumption, comprising the lipid composition of the invention, wherein the DHA and the second and third polyunsaturated fatty acids are provided in the form of triglyceride esters is provided. Will be done. Ethyl esters are particularly suitable for use in dietary supplements because these ester forms can be produced efficiently and easily and do not require conversion to triglyceride forms. Therefore, in a further embodiment, the DHA and the second and third PUFAs are provided independently, either in the form of fatty acid ethyl ester or as part of the triglyceride.

Triglycerides are esters derived from glycerol and three fatty acids. Since the present invention relates to a blend of fatty acids, the fatty acid components in such triglycerides can be mixed in corresponding proportions. That is, a mixture of different triglyceride molecules may be present in the composition, but the overall fatty acid profile in the composition is as defined in the claims.

Alternatively, the fatty acid component may be present in the form of a "free" fatty acid, i.e. the -COOH form of the fatty acid. However, in certain compositions of the invention, this form of fatty acid is relatively free because the composition has an unpleasant (often "soap-like") taste and is less stable than the esterified form of the fatty acid. Contains at low levels. Free fatty acids are typically removed from the oil and lipid compositions by alkali or physical purification, eg, according to the processes discussed elsewhere herein. Thus, in one embodiment, the total free fatty acid content in the lipid composition is less than 5% by weight (eg, less than 3% by weight, especially less than 2% by weight) of the total fatty acid content of the composition.

The fatty acids in the lipid compositions of the present invention are typically linear (ie, unbranched) chain fatty acids. The compositions of the invention that may be mentioned include those containing very low levels of branched chain fatty acids and esters thereof such that the composition is essentially free of branched chain fatty acids and branched chain fatty acid esters. The term "low level" means that the composition contains branched chain fatty acids and fatty acid esters in an amount of up to about 0.1% by weight of the total fatty acids of the composition.

The lipid compositions of the present invention may also contain other components (eg, other than fatty acids) that are derived from the raw material and are not completely removed during the extraction and concentration processes. The exact identity of these other ingredients varies widely from material to material. Examples of such other components are as free sterols or as sterol esters (β-citosterol, β-citostanol, Δ5-avenasterol, campesterol, Δ5-stigmasterol, Δ7-stigmasterol and Δ7-. Includes plant sterols (ie, plant sterols and plant stanols) that are present either as avenasterols, cholesterol, brush casterols, carinasterols, campesterols, campesterols and evericols. Other examples include antioxidants such as tocopherols and tocotrienols. Thus, certain lipid compositions of the invention that may be mentioned include those containing a detectable amount of one or more phytosterols (eg, β-sitosterol). Such sterols can be present in at least about 0.01% by weight, but typically no more than about 1% by weight, of the lipid composition.

The compositions of the present invention are advantageously available from plant sources (“vegetable” sources). The term "vegetable-based" means that at least 70% by weight of the lipids present in the compositions of the present invention are obtained from the vegetable source. Vegetable sources include plant sources, especially crops such as grains. In at least one embodiment, the lipids are obtained from seed oil crops of the genus Brassica, such as Brassica napus or Brassica juncea. However, to avoid misunderstanding, it is not essential that the composition be obtained only from such sources, i.e. some of the lipids in the compositions of the invention (eg, up to 30% by weight) are in the ocean. It may be obtained from other sources, including (eg, fish or crustacean) oils, algal oils, and combinations thereof. In one example, at least 80% by weight, for example at least 90% by weight, of the lipids present are obtained from the plant source. In a particular composition of the invention, essentially all (ie, at least 95%, at least 99%, or about 100%) lipids are obtained from plant sources.

In one embodiment, the compositions of the invention (and the feed and pharmaceutical compositions as defined herein below) are not of animal (eg, marine animal) origin. That is, in such an embodiment, the lipid composition does not contain any components derived from animals such as fish and crustaceans. Lipid compositions in which no components are obtained from animals are considered advantageous in terms of lipid content and stability profile that can be achieved according to standard purification and / or concentration procedures.

There are many advantages to using plants as a source of lipids or fatty acids. For example, marine oil sources are known to contain relatively high levels of pollutants not found in plant materials, such as mercury, PCBs, and fish allergens (eg, parvalbumin). .. Historical overfishing has also depleted fish and crustacean stocks (eg krill) and is no longer sustainable. Accordingly, the present invention provides a sustainable source of polyunsaturated fatty acid oil compositions containing relatively low levels of unwanted contaminants.

In certain embodiments, the compositions of the invention are derived from plants. The oily plants are typically oilseed crops such as copra, cotton seeds, flax, palm kernels, peanuts, rapeseed, soybeans, and sunflower seeds. Therefore, compositions obtained only from plants are sometimes referred to as "vegetable" oils or "vegetable lipid compositions". Suitable plants from which the lipid compositions of the present invention can be obtained are known to those of skill in the art, and Brassica sp. , Gossypium hersutum, Linum usitatissimum, Helianthus sp. , Cornamus tinctorius, Glycine max, Zea mays, Arabidopsis thaliana, Sorghum bicolor, Sorghum vulgare, Avena sativa, Trifoliaum sp. , Elaesis guineenis, Nicotiana benthamiana, Hordeum vulgare, Lupinus angustifolia, Oryza sativa, Oryza graberrima, Camelina sativa, or Camelina sativa. Specific plant sources that may be mentioned in this regard are Brassica sp. Is.

Suitable sources, including marine, algae, and plant sources, can be naturally occurring or genetically modified to enhance their ability to produce long-chain polyunsaturated fatty acids. Examples of plant sources genetically modified for this purpose, i.e. derived from recombinant plant cells, are known to those of skill in the art and International Patent Application No. PCT / AU2013 / 000639 (published as WO2013 / 185184). It is disclosed in the same PCT / AU2014 / 050433 (published as WO2015 / 089587) and the same PCT / AU2015 / 050340 (published as WO2015 / 196250). Genetically modified canolas are described in WO2017 / 218969 and WO2017 / 219006. The disclosures in all publications described herein are incorporated by reference in their entirety.

The lipid compositions of the present invention can be obtained directly from naturally occurring sources (eg, animals, algae, and / or plants). However, they typically need to be processed to concentrate the oils obtained from naturally occurring sources. Suitable enrichment processes are illustrated in the Examples.

Suitable sources of the lipid compositions of the present invention, or "crude" oils that can be blended or concentrated to produce those compositions, include marine species, algae and plants. The process for obtaining oil from marine sources is well known in the art.

As discussed above, plant sources (such as oil seed sources) are particularly suitable due to their low levels of certain pollutants and their high sustainability. Brassica sp. Plants such as (eg, canola) produce seeds that can be processed to obtain oil.

Oil / Lipid Extraction Oils produced by plants and seeds can be extracted, processed, and analyzed using techniques routinely practiced in the art. Typically, plant seeds are cooked, squeezed, oiled and produced crude oil. The oil can then be degummed, refined, bleached, and / or deodorized. A combination of degumming, purification, bleaching, and deodorization has been found to be particularly effective in preparing a lipid mixture enriched with LC-PUFA. Thus, in one embodiment, the lipid composition is obtained from degummed, purified, bleached, and / or deodorized seed oils. However, it is not necessary to process the oil in this way and proper purification and concentration can be achieved without using these methods.

Generally, techniques for crushing seeds are known in the art. For example, oilseed seeds are tempered by spraying them with water and increasing the moisture content to, for example, 8.5%, and flaking using a smooth roller with a gap setting of 0.23 mm to 0.27 mm. can do. Depending on the type of seed, water may not be added before crushing. Extraction can also be achieved using an extrusion process. The extrusion process may or may not be used in place of flaking and may be used as an add-on process either before or after the screw press.

In one embodiment, most of the seed oil is released by grinding using a screw press. The solids discharged from the screw press are then extracted with a solvent, such as hexane, using a heat trace column, after which the solvent is removed from the extracted oil. Alternatively, the crude oil produced by the press operation can be passed through a settling tank provided with a slotted wire drainage top to remove solids expressed in the oil during the press operation. The clarified oil can pass through the plate and frame filters to remove any residual fine solid particles. If desired, the oil recovered in the extraction process can be combined with the clarified oil to produce a mixed crude oil. Once the solvent was removed from the crude oil, the squeezed and extracted parts were combined and subjected to normal petroleum processing procedures.

Purification and Purification As used herein, the term "purified" as used in connection with the lipids or oils of the invention typically means that the extracted lipids or oils are lipids /. It means that it has been subjected to one or more processing steps to increase the purity of the oil component. For example, the purification step may include one or more of degumming, deodorizing, decoloring, or drying the extracted oil. However, as used herein, the term "purified" refers to transesterification processes, or lipids or oils of the invention, to increase LC-PUFA content as a percentage of total fatty acid content. Does not include another process of altering the fatty acid composition of. In other words, the fatty acid composition of the purified lipid or oil is essentially the same as the fatty acid composition of the unpurified lipid or oil.

Vegetable oils can be refined (purified) once extracted from the plant source using one or more of the following processes, especially using a combination of degumming, alkaline refining, bleaching and deodorizing. Suitable methods are known to those of skill in the art (eg, those disclosed in WO 2013/185184).

Degumming is an early stage of oil refining and its main purpose is to remove most of the phospholipids from the oil. Typically, addition of about 2% water containing phosphoric acid to the crude oil at 70-80 ° C. results in the separation of most phospholipids with trace amounts of metals and pigments. The insoluble material removed is primarily a mixture of phospholipids and triacylglycerols. Degaming can be performed by adding concentrated phosphoric acid to crude seed oil, converting non-hydrated phospholipids into hydrateable forms, and chelating the rare metals present. The gum is separated from the seed oil by centrifugation.

Alkaline refining is one of the refining processes for processing crude oil and is sometimes referred to as neutralization. It usually follows degumming and precedes bleaching. Following degaming, the seed oil can be treated by adding a sufficient amount of alkaline solution to titrate all free fatty acids and phosphoric acid and removing the soap thus formed. Suitable alkaline materials include sodium hydroxide, potassium hydroxide, sodium carbonate, lithium hydroxide, calcium hydroxide, calcium carbonate, and ammonium hydroxide. Alkaline purification is typically performed at room temperature to remove the free fatty acid fraction. The soap is removed by centrifugation or extraction of the soap into a solvent and the neutralized oil is washed with water. If desired, any excess alkali in the oil can be neutralized with a suitable acid such as hydrochloric acid or sulfuric acid.

Bleaching is performed by operating the oil in the presence of bleached soil (0.2-2.0%) and in the absence of oxygen, with nitrogen or steam, or in vacuum, at 90-120 ° C. 10-30. It is a purification process that heats for minutes. Bleaching is designed to remove unwanted pigments (carotenoids, chlorophyll, etc.), and the process also removes oxidation products, trace metals, sulfur compounds, and traces of soap.

Deodorization is the treatment of fats and oils at high temperatures (eg, about 180 ° C.) and low pressure (0.1 to 1 mm Hg). This is typically achieved by introducing steam into the seed oil at a rate of about 0.1 ml / min / 100 ml seed oil. Approximately 30 minutes after spraying, the seed oil is cooled under vacuum. This treatment improves the color of the seed oil and removes most of the volatile or odorous compounds, including any residual free fatty acids, monoacylglycerols, and oxidation products.

Dewaxing is a process sometimes used in the commercial production of oils to separate fats and oils into solid (stearin) and liquid (olein) fractions by crystallization below ambient temperature. Originally applied to cottonseed oil, it produced solid-free products. It is typically used to reduce the saturated fatty acid content of oils.

Transesterification Crude oil usually contains the fatty acid of interest in the form of triacylglycerol (TAG). Transesterification is a process that can be used to exchange fatty acids within and between TAGs, or to transfer fatty acids to another alcohol to form an ester (such as an ethyl ester or methyl ester). In embodiments of the invention, transesterification is typically achieved using chemical means containing strong acids or bases as catalysts. Sodium ethoxide (in ethanol) is an example of a strong base used to form fatty acid ethyl esters by transesterification. This process can be carried out at ambient temperature or high temperature (eg, up to about 80 ° C.).

In other embodiments of the invention, transesterification is accomplished using one or more enzymes, particularly lipases known to be useful for hydrolyzing ester bonds in, for example, glycerides. NS. The enzyme can be a lipase that is position-specific (specific to sn-1 / 3 or sn-2) to the fatty acids on the triacylglycerides or that prefers some fatty acids over others. .. Specific enzymes that may be mentioned include Lipozyme 435 (available from Novozymes A / S). This process is typically carried out at ambient temperature. This process is typically carried out in the presence of an excess amount of alcohol corresponding to the desired ester form (eg, by using ethanol to form the ethyl ester of the fatty acid).

Distillation Molecular distillation is an effective method for removing large amounts of highly volatile components such as saturated fatty acids from crude oil. Distillation is typically carried out under reduced pressure, for example less than about 1 mbar. The temperature and time can then be selected to achieve an approximately 50:50 split between the distillation and the residue after a distillation time of several hours (eg, 1-10). Typical distillation temperatures used to produce the lipid compositions of the present invention range from 120 ° C to 180 ° C, especially 145 ° C to 160 ° C.

Multiple distillations can be performed and each distillation is considered complete when an approximately 50:50 division between the distillation and the residue is achieved. The use of continuous distillation reduces the overall yield, but two distillations may give optimum results.

Chromatography Chromatography is an effective method for separating the various components of an LC-PUFA mixture. It can be used to increase the concentration of one or more preferred LC-PUFAs in the mixture. Chromatographic separation can be achieved under a variety of conditions, but typically involves the use of a fixed bed chromatography system or a simulated moving bed system.

In a fixed bed chromatography system, a mixture with components to be separated permeates a column (stationary phase) containing a packing of a porous material that is highly permeable to fluids (usually with an eluent). Based on the concept of letting. The permeation rate of each component of the mixture depends on the physical properties of that component so that the component is continuously and selectively ejected from the column. Thus, some of the components will tend to be more delayed as they tend to lock strongly into the stationary phase, while others will tend to lock weakly and drain from the column after some time.

The simulated moving bed system consisted of several separate columns containing adsorbents, which were connected in series together and the mixture and eluent injection points in the system, and separated. The component collection points are also manipulated by periodically shifting, so that the holistic action simulates the operation of a single column containing a moving bed of solid adsorbent. Thus, the simulated moving bed system, like a conventional fixed bed system, consists of a column containing a fixed bed of solid adsorbent through which the eluent passes, but in the simulated moving bed system, The operation is like simulating a continuous countercurrent moving floor.

The columns used in these processes typically contain silica (or modified silica) as the basis for the stationary phase. The mobile phase (eluent) is typically a highly polar solvent mixture, often containing one or more protic solvents such as water, methanol, ethanol, and mixtures thereof. The eluent flow rate can be adjusted by one of ordinary skill in the art to optimize the efficiency of the separation process. For example, the products defined in the claims can be obtained using a relatively fast eluent flow rate. Using a slower flow rate can improve the separation of PUFAs contained in the initial mixture and thus increase the concentration of DHA. Detection methods for LC-PUFA are known to those of skill in the art and include UV-vis absorption methods as well as refractive index detection methods.

Therefore, according to the second aspect of the present invention, a process for producing the lipid composition of the present invention is provided, in which the process provides a mixture of fatty acid ethyl esters and the mixture is subjected to a chromatographic separation process. Including to serve. The present invention also relates to lipid compositions obtained by such a process. Suitable chromatographic separation conditions include those described herein.

For example, a particular mobile phase that can be used in chromatographic separation is a mixture of methanol and water (eg, 88% methanol / water), which is used during the separation process to improve efficiency. Can be modified (eg, to increase methanol content). A particular stationary phase that can be used is a silica-based stationary phase such as a Deltaprep C18 column. Analytical HPLC, or other suitable technique known to those of skill in the art, has been performed on the resulting fractions to contain a sufficiently high concentration of the desired PUFA, thus providing the lipid compositions of the present invention. The contained fraction can be identified.

In an embodiment of a second aspect of the invention, the mixture of fatty acid ethyl esters is obtained, for example, by transesterification and distillation of vegetable-based lipid oils according to any one of the processes described herein. Be done. Plant-based lipid oils can be obtained from any plant disclosed herein or known in the art, especially oil seeds. Prior to transesterification and distillation, purification of vegetable-based lipid oils using degumming, alkaline purification, bleaching, and / or deodorization can optionally be performed.

Other Concentration Methods The lipid compositions of the present invention are useful as pharmaceutical active ingredients (APIs) or as precursors (or "intermediates") of APIs that can be obtained from them by further concentration. Such compositions are further concentrated at levels of beneficial PUFAs such as DHA and / or ALA.

Concentrations of polyunsaturated fatty acids in oils are increased by various methods known in the art, such as freeze crystallization, complexing with urea, supercritical fluid extraction and silver ion complexing. be able to. Complexing with urea is a simple and efficient way to reduce the levels of saturated and monounsaturated fatty acids in oils. First, the TAG of an oil is divided into their constituent fatty acids, often in the form of fatty acid esters. These free fatty acids or fatty acid esters can then be mixed with an ethanol solution of urea for complex formation, as the treatment usually does not change the fatty acid composition. Saturated and monounsaturated fatty acids can be easily complexed with urea, crystallized upon cooling and then removed by filtration. As a result, the non-urea complex fraction is fortified with long-chain polyunsaturated fatty acids.

Product The lipid composition of the present invention is a bulk oil. That is, the lipid composition is separated from the raw material from which some or all of the lipid was obtained (eg, plant seeds).

The lipid composition of the present invention can be used as a feed. That is, the compositions of the present invention may be provided in a form that can be used orally. For the purposes of the present invention, a "feed", when taken into the body, helps to nourish or build tissue, or provide energy, and / or proper nutritional status or Includes any food or preparation for human consumption that maintains, restores, or supports metabolic function. Feeds include nutritional compositions for babies and / or toddlers, such as infant formulas. In the case of feed, fatty acids can be provided in the form of triglycerides to further minimize unpleasant taste and maximize stability.

The feed comprises the lipid composition of the present invention with an optional suitable carrier. The term "carrier" is used in its broadest sense and includes any ingredient that may or may not have nutritional value. As will be appreciated by those skilled in the art, the carrier should be suitable for use in the feed (or used in a sufficiently low concentration) so as not to have a harmful effect on the organism consuming the feed.

The feed composition can be in solid or liquid form. In addition, the composition may contain the desired amount of edible macronutrients, proteins, carbohydrates, vitamins, and / or minerals for a particular application, as is well known in the art. The amount of these ingredients depends on whether the composition is intended for use in normal individuals or in individuals with special needs, such as individuals suffering from metabolic disorders.

Examples of suitable nutritious carriers include edible fats (eg, coconut oil, borage oil, fungal oil, black tide oil, soybean oil, and monoglycerides and diglycerides), carbohydrates (eg, glucose, edible lactose, and hydrolysis). Includes major nutrients such as starches) and proteins (eg, soybean proteins, electrodialysis whey, electrodialysis skim milk, milk whey, or hydrolyzates of these proteins).

Vitamins and minerals that can be added to the feeds disclosed herein include, for example, calcium, phosphorus, potassium, sodium, chloride, magnesium, manganese, iron, copper, zinc, selenium, iodine, and vitamins A, E. , D, C and B complexes are included.

The lipid composition of the present invention can be used in a pharmaceutical composition. Such pharmaceutical compositions optionally include the lipid compositions of the invention, along with one or more pharmaceutically acceptable excipients, diluents or carriers known to those of skill in the art. Suitable excipients, diluents or carriers include phosphate buffered saline, water, ethanol, polyols, wetting agents, or emulsions such as water / oil emulsions. The composition can be in either liquid or solid form, including solutions, suspensions, emulsions, oils or powders. For example, the composition can be in the form of tablets, capsules, encapsulated gels, ingestible liquids (including oils or solutions) or powders, emulsions, or topical ointments or creams. The pharmaceutical composition may also be provided as an intravenous formulation.

Specific forms suitable for feed and pharmaceutical compositions include liquid-containing capsules and encapsulated gels.

The lipid compositions of the present invention can be mixed with other lipids or lipid mixtures, especially plant-based fatty acid esters and fatty acid ester mixtures, prior to use. The lipid compositions of the present invention, along with one or more additional ingredients selected from the group consisting of antioxidants (eg, tocopherols (eg alpha-tocopherols or gamma-tocopherols) or tocotrienols), stabilizers, and surfactants. Can be provided. Both alpha-tocopherol and gamma-tocopherol are naturally occurring components of various vegetable seed oils, including canola oil.

For example, it may be desirable to include isotonic agents such as sugars and sodium chloride. In addition to such Inactive Diluents, the composition can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening agents, flavoring agents and aromatics. In addition to the lipid compositions of the invention, suspensions include ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, metahydroxyaluminum, bentonite, agar, and tragacanth or mixtures thereof. Can include suspending agents such as.

Solid dosage forms such as tablets and capsules can be prepared using techniques well known in the art. For example, fatty acids produced according to the methods disclosed herein are combined with binders such as acacia, cornstarch or gelatin, disintegrants such as potato starch or alginic acid, and lubricants such as stearic acid or magnesium stearate. , Lactose, sucrose, and cornstarch can be tableted on a conventional tablet basis. Capsules can be prepared by incorporating these excipients into gelatin capsules with the relevant lipid composition and optionally one or more antioxidants.

Possible routes of administration of the pharmaceutical compositions of the present invention include, for example, enteral (eg, oral and rectal) and parenteral. For example, the liquid preparation can be administered orally or transrectally. Additionally, the homogeneous mixture can be completely dispersed in water and mixed with physiologically acceptable diluents, preservatives, buffers, or propellants under sterile conditions to form sprays or inhalants. can.

The lipid compositions of the present invention are shown as pharmaceuticals. According to a further aspect of the invention, there is provided a composition of the invention comprising any of the above pharmaceutical compositions for use as a pharmaceutical product.

The lipid compositions of the present invention can typically provide many advantages associated with long chain polyunsaturated fatty acids. For example, the lipid compositions of the invention, and the pharmaceutical compositions described above, can be determined using tests well known to those skilled in the art, such as treatment or prevention of cardiovascular disease, protection against death of patients with cardiovascular disease, overall. Can be used in lowering serum cholesterol levels, lowering hypertension, increasing HDL: LDL ratios, lowering triglycerides, or lowering apolipoprotein B levels. Therefore, methods of treating (or preventing) the diseases and conditions listed above using the lipid compositions of the present invention are also disclosed.

As used herein, the terms "treatment," "treat," and "treating" are compared to what would occur in the absence of measures to be taken. Thus, reversing, alleviating, or inhibiting the progression of the disease or disorder described herein, or delaying, eliminating, or reducing the incidence or onset of the disorder or disease as described herein. Point to. As used herein, the terms "prevent", "prevention" and "preventing" refer to reducing the risk of acquiring or developing a given condition, or illness. Refers to recurrence or reduction or inhibition of the condition in a non-subject.

A typical dose of a particular fatty acid is 0.1 mg to 20 g and is ingested once to 5 times a day (up to 100 g a day), in particular about 10 mg to about 1, 2, 5 or a day. It is in the range of 10 g (taken in one or more doses). As is known in the art, at least about 300 mg / day of fatty acids, especially LC-PUFAs, are desirable. However, it will be appreciated that any amount of fatty acid is beneficial to the subject.

When used as a pharmaceutical composition, the dose of the lipid composition administered to the patient is determined by one of the conventional techniques in the art, eg, patient weight, patient age, patient overall. It depends on various factors such as good health, the patient's past medical history, and the patient's immune status.

The compositions of the present invention are readily available compositions, which may have an improved stability profile, the relative ratio of omega-3 to omega-6 fatty acids being particularly beneficial to human health. It may contain a mixture of certain fatty acids. Stability can be assessed using a variety of methods known to those of skill in the art. Such methods include the Lansimat method, evaluation of propanal formation (particularly suitable for omega-3 fatty acids), evaluation of hexanal formation (particularly suitable for omega-6 fatty acids), "peroxide value". Methods (eg, using the AOCS formula method Cd8-53) and the "p-anisidine value" method (eg, using the AOCS formula method Cd18-90) are included. The compositions of the present invention have an improved stability profile compared to reference blends, which have similar compositions for the major LC-PUFAs but contain significant amounts of animal (fish) -derived lipids. It is shown in the examples that it is available from a starting mixture that does not show.

The compositions of the present invention are more effective, less toxic, longer lasting, more effective, less side effects, and more easily absorbed than the lipid compositions known in the prior art. , And / or have a better pharmacokinetic profile (eg, higher oral bioavailability and / or lower clearance), and / or have other useful pharmacological, physical, or chemical properties. It may also have the advantage of obtaining.

The present invention will be described with reference to the following examples.

Propanal release data for canola oils and reference oils (after transesterification, distillation, and chromatography) are presented to demonstrate improved stability of the canola oils described herein.

Propanal release data for canola oils and reference oils (after RBD purification, ester exchange, distillation, and chromatography) are presented to demonstrate improved stability of the canola oils described herein.

Example 1-Extraction of DHA canola oil from seeds The varieties of canola disclosed in US Patent Publication No. US2018 / 0016590A1 were cultivated as summer crops. After harvesting the seeds, they were stored at room temperature before crushing.

A Kern Kraft KK80 screw press was used to grind 272 kg of seeds to produce DHA oil. The temperature of the expeller color heater was set to the maximum set temperature of the thermostat. The initial ambient temperature and choke temperature were 20 ° C., and the choke distance was set to 73.92 mm. The seeds were fed with continuous collection of oil and coarsely ground flour without stopping the expeller until all seeds were ground.

The rotation speed of the auger, the temperature of the coarse grind and the effluent were monitored throughout the press. The crushing time, which is a throughput rate of 67.5 kg / hour, was 270 kg for 4 hours. A yield of 87.2 kg of crude oil (32%) was obtained. After filtration to remove fines, the yield was 77.2 kg (28%).

Example 2-Reference Blend Oil Pure fish oil contains low levels of ALA fatty acids, as well as significantly higher levels of EPA and DHA. The reference oil blend (referred to herein as "crude triglyceride reference blend oil" or the like) was designed to be as similar in composition as possible to the filtered DHA canola oil obtained in Example 1. .. This was done by (a) matching the total level of DHA with the level of DHA canola oil and (b) matching the ratio of DHA / (ALA + EPA). This was achieved by blending DHA (tuna) -rich fish oil, ALA (linseed oil) -rich oil, and standard canola oil. The resulting reference blend oil also has a total omega-3 content similar to that of DHA canola oil.

Example 3-Fatty Acid Composition of Crude DHA Canola Oil and Reference Blend The fatty acid composition of the filtered crude oil and the reference blend oil was analyzed. The results are shown below.

Example 4-Oil Stability Assessment Lansimat stability studies were performed using the crude DHA canola oils and reference blends described in Example 1 and Example 2, respectively. This method involved testing about 2.5 g of test material using the standard procedure of Metrohm743 Ranchimat at 90 ° C.

ＤＨＡキャノーラ油は、参照油よりも一貫して劣った安定性を示した。 The table below summarizes the results obtained from these oils at 90 ° C. The experiment was carried out twice.

DHA canola oil showed consistently inferior stability over the reference oil.

Example 5-Enzymatic transesterification of crude canola-DHA oil The following enzymatic transesterification procedure was performed on about 5 kg of crude triglyceride oil obtained in Example 1. Lipozymes 435 was obtained from Novozymes A / S.

To a dry nitrogen flush reactor equipped with a mechanical stirrer, 100% undenatured ethanol (5.00 kg) and the crude triglyceride oil obtained in Example 1 (5.00 kg) were added and the mixture was stirred. Lipozyme 435 (420 g) was added to the mixture and the mixture was heated at 40 ° C. for 21 hours. ^{The recorded 1} H NMR spectrum of the sample taken from the mixture showed that the reaction was complete.

The mixture was cooled to 20 ° C. The mixture was discharged from the reactor and filtered through a 4 μm polypropylene filter cloth on a 20 L cheesecloth. The reactor was rinsed with ethanol (2 x 1.6 L) and petroleum spirit (2.51), which were used to sequentially wash the filter cake. Petroleum spirit (10 L) and water (4 L) were added to the resulting crude reaction mixture, the mixture was thoroughly mixed in the reactor and then allowed to stand, after which two phases were formed.

The petroleum spirit layer was removed and the aqueous layer was further extracted with petroleum spirit (2 x 10 L). The combined petroleum spirit layer was returned to the reactor and evaporated to a small amount (about 10 L) in vacuo. The resulting concentrated solution was drained from the reactor, dried over anhydrous magnesium sulfate (about 1 kg), filtered and concentrated in vacuo to give a yellow oil (yield: 99%).

Example 6-Enzymatic transesterification of crude reference blend oil Enzymatic transesterification of crude triglyceride reference blend oil (5.00 kg) obtained according to Example 2 uses the process described in the previous example. Completed. The product was obtained as a yellow oil.

Example 7-Distillation of transesterified canola oil Standard procedure for removing more volatile components of fatty acid ethyl ester (FAEE) mixture by vacuum distillation

The crude fatty acid ethyl ester (FAEE) from crude canola-DHA (obtained in Example 5) was subjected to distillation under the following conditions. Separation by distillation was accomplished by passing transesterified crude oil through a Pope 2 inch (50 mm) thin film still under vacuum with a 2x1000 ml collection flask for collecting the distillate and residue. The composition of each fatty acid was analyzed.

The vacuum was supplied by the Edwards 3 rotary pump and the vacuum was measured by the ebro pressure gauge VM2000.

The oil was fed to the distiller at 4 mL / min by the Cole-Palmer Instrument Company's easy-load II peristaltic pump, the distiller motor was set to 325 rpm and the distillate was condensed using a water condenser. Supply continued until one of the receiver flasks was full.

The crude canola DHA FAEE was distilled under these conditions with the heater band initially set at 147 ° C. The purpose was to obtain a 50:50 split of Distillate: Residue. During the first 30-45 minutes of the experiment, the temperature of the heater band was raised to 154 ° C. to increase the proportion of distilled oil and then the distiller was equilibrated. After 30 minutes, the temperature of the heater band was adjusted to 149 ° C. over 30 minutes. The remaining distillation was carried out at 149 ° C. The total distillation time was 350 minutes. By distillation under standard conditions where the temperature of the heater band was set to 149 ° C., some of the residue from the above distillation was again subjected to the removal of the more volatile components. The total distillation time was 95 minutes.

Example 8-Distillation of FAEE from transesterified crude reference blend The crude fatty acid ethyl ester (FAEE) from the reference crude blend (obtained in Example 6) is the same as shown in the previous example. It was subjected to distillation under the conditions.

The reference crude blend FAEE was distilled under these standard conditions with the heater band initially set at 152 ° C. The purpose was to obtain a 50:50 split of Distillate: Residue. After 20 minutes, the temperature of the heater band was set to 154 ° C. to increase the flow of the distillate. After another hour, the temperature of the heater band was adjusted to 153 ° C. and to 152 ° C. in the next hour. The temperature of the heater band was set to 153 ° C. for the last hour of distillation. The total distillation time was 380 minutes. The residue from the above distillation was resubmitted to removal of the more volatile components by distillation under standard conditions. The purpose was to obtain a 50:50 split of Distillate: Residue. Distillation was mainly carried out with the heater band set to 150-151 ° C. The total distillation time was 195 minutes.

Example 9-Chromatographic Separation Separation HPLC of FAEE from Canola
The fatty acid ethyl ester (FAEE) obtained in Example 7 (ie, obtained from crude canola-DHA and treated using transesterification and distillation) was subjected to chromatographic separation under the following conditions: A preparative HPLC system with a Waters Prep 4000 system, a 10 ml loop Rheodyne injector, a 300 x 40 mm Deltaprep C18 column, a Waters2487 dual wavelength detector, and a chart recorder at 70 mL / min in 88% methanol / water mobile phase. Equilibrated. The detector was set to full scale in 215 nm and 2.0 absorbance units and charts were run at 6 cm / hour.

1.0 g of FAEE oil was dissolved in a minimum amount of 88% methanol / water and injected into the column via a Rheodyne injector. When the solvent front appeared after about 7 minutes, about 250 mL fractions were collected.

Analytical HPLC was performed on all fractions and combined "symmetrical" fractions predominantly containing DHA (yield: 28%).

Analytical HPLC
For sample analysis, an HPLC system equipped with a Waters 600E pump controller, 717 autosampler, 2996 photodiode array detector, and 2414 refractometer detector was used. The analysis was performed on a 150 x 4.6 mm Alltima C18 column using either isocratic 90% methanol / water or 95% methanol / water as the mobile phase at 1.0 mL / min. .. Data collection and processing was performed with Waters Emper3 software.

Chromatographic Separation of FAEE from Example 10-Reference Blend Distilled fatty acid ethyl ester (FAEE) of the reference blend (obtained in Example 8) is chromatographed under the same conditions as shown in the previous example. It was used for grafography separation.

Analytical HPLC was performed on all fractions, combining "symmetrical" fractions predominantly containing DHA.

Example 11-Fatty acid composition analysis of concentrated oil The fatty acid composition of the products obtained in Examples 9 and 10 was analyzed. The results are shown below.

Example 12-Oil Stability Assessment Headspace GC-MS Stability Test Headspace analysis was performed on the above concentrated products to assess the amount of propanal released under certain conditions. Increased levels of propanal release indicate reduced stability of the test material.
The SPME (Solid Phase Microextraction) Method 65 μm PDMS / DVB TableFlex Fiber (Supelco Fiber Kit 57284-u) was selected.
The fibers were conditioned in a Triples RSH conditioning station for 10 minutes before use at 250 ° C.
Samples were incubated at 40 ° C. for 1 minute prior to extraction.
Extracted from the Headspace vial for 1 minute.
It is expected to be an excellent general method capable of capturing a wide range of volatile components.
GC method Thermo Scientific TRACE1310 GC
Thermo Scientific TR-DIOXIN 5MS column, inner diameter 0.25 mm, 30 m film 0.1 μm
250 ° C division 83, 1.2 ml He / min GC lamp: 40 ° C at 5 ° C / min, 1 min to 100, then up to 300 ° C at 50 ° C / min.

A common MS-specific column was used that showed excellent synergies for headspace analysis. A slow initial temperature rise was employed to maximize the separation of volatiles before maximizing to maintain column performance. Divided injection was adopted to avoid the need for cryogenic cooling of the inlet and improve the separability of the column.

Standard separation was hampered by peak overlap, but was still quantifiable. Three standard calibration results (0.1, 0.01, and 0.01%), molecular ion m / z56, were used to detect propanal. The m / z 58 base peak is used to detect hexanal.
MS Method Thermo Scientific DFS High Resolution GC-MS
Low resolution (1000), full scan 35-350 Da, 0.5 sec / scan standard: -Standard dilutions of propanal and hexanal were made on the supplied DHA canola ethyl ester. These standard mixtures were then added to 20 ml headspace vials in a volume of 540 μl.

Full scan has been adopted, making it possible to monitor all developed products, not specific molecules.

ＤＨＡキャノーラ油は、参照油と比較して酸化に対して優れた安定性を示した。 Headspace stability results:
The table below summarizes the results obtained from the canola oil obtained in Example 9 and the reference oil obtained in Example 10 at T = 0-5 days. The test sample was kept at ambient temperature during this period, on a light box and under fluorescent tube illumination. Analyzing the m / z 58 molecular ions, the mass chromatogram clearly shows the appearance of propanal at 1.37 minutes at room temperature. The table below quantifies the evolution of propanal and the data are shown in Figure 1. DHA canola oil releases substantially a small amount of propanal, demonstrating that the stability of canola oil is improved compared to the reference.

DHA canola oil showed superior stability to oxidation compared to reference oil.

Purification of Example 13-DHA Canola Oil A portion of the canola oil obtained in Example 1 was refined before undergoing further concentration. Purification processes included degumming, alkaline purification, bleaching, and deodorization.

Acid degumming Degumming is the removal of non-hydrating and hydrating phospholipids from oils. The dry crude oil obtained in Example 1 was heated to 53 ± 2 ° C., and a 0.2% 50% citric acid solution was added. After mixing for about 30 minutes, 2.0% heated (53 ± 2 ° C.) soft water was added and mixed for about 30 minutes. The oil was heated to 67 ± 3 ° C. during retention and then centrifuged.

Acid pretreatment / purification Purification is the removal of free fatty acids after saponification with a corrosive agent to make them water-soluble, followed by centrifugation. An acid pretreatment step was used to continue hydration of phosphatide. The degumming oil was heated to 65 ± 5 ° C., 0.1% 85% phosphoric acid was added and mixed for a total of 30 minutes. After acid addition and retention time, 20 Be'(Baume; 14.4%, w / w) sodium hydroxide was added to neutralize free fatty acids and 0.05% (w / w) excess. The corrosive and oil were then mixed for an additional 15 minutes. The oil was heated to 62 ± 2 ° C. while holding for 15 minutes, and then the oil was centrifuged.

Trisyl Silica Treatment To further remove the soap, Trisyl silica treatment was performed to a level compatible with bleaching. Trisyl pretreatment was combined with a bleaching step. The refined oil was heated to 68 ± 5 ° C. and treated with 0.3% Trisyl 300. After mixing the oil / Trisyl for about 15 minutes, bleaching was continued.

The bleached refined oil was treated with adsorptive clay to remove peroxides, phospholipids, color bodies, and trace amounts of soap. An acid pretreatment step was used to continue hydration of phosphatide. Trisyl pretreatment oil was mixed with a 0.2% (w / w) 50% citric acid solution. After mixing for 15 minutes, 2% (w / w) Tonsil Supreme 126FF bleached clay was added. The mixture was then heated to 90 ± 2 ° C. under vacuum and held for about 30 minutes. The oil was cooled to 60 ± 2 ° C., evacuated with nitrogen and filtered with 1.0 kg of filtration aid. Pressure vessel: All 316 stainless steel construction with 500 L Cherry-Burrel pressure vessel, steam or cooling water jacket, impeller and baffle for mixing, serial number E-227-94. Filter press: 24 ″ Polypropylene Superior Filter Press, 4.8 cu capacity ft filter, paper and cloth support were used.

The deodorized and bleached oil was applied with steam at high temperature and low pressure to remove odorous components, flavor components and additional free fatty acids. The color also fades due to heat bleaching at high temperatures. Half of the bleaching oil was deodorized with 1% spurge steam at 180 ± 2 ° C. for 60 minutes and the fatty acid composition (FAC) was monitored. Deodorizing vessel (OD4): 400L Coppersmithing vacuum rated vessel, steam or antifreeze jacket, all 316 stainless steel construction. A slight decrease in DHA levels was observed after holding at 180 ° C. for 60 minutes. Another test was then performed by holding at 180 ° C. for 30 minutes. The product was packed in a 20 L plastic HDPE pail under nitrogen and stored in a 4 ° C cooler.

Example 14-Refining the original reference blend oil Some of the reference blends described in Example 2 were refined before further concentration. In the purification process, the reference blend was purified under the same conditions as shown in the previous example.

Fatty Acid Composition of Crude DHA Canola Oil of Example 15-RBD and Reference Blend The fatty acid composition of the filtered crude oil of RBD (of Example 13) and the RBD reference blend oil (of Example 14) was analyzed. The results are shown below.

References to "RBD" related to Examples (and attached figures) are that the product in question is "refined" in either Example 13 (for canola oil) or Example 14 (for reference blends). It means that it was obtained directly or indirectly from the "made" product.

Enzymatic transesterification of Example 16-RBD canola-DHA oil Enzymatic transesterification of the crude triglyceride reference blend oil (5 kg) obtained according to Example 13 was completed using the process described in Example 5. .. The product was obtained as a yellow oil.

Enzymatic transesterification of Example 17-RBD reference blend oil Enzymatic transesterification of the crude triglyceride reference blend oil (5 kg) obtained according to Example 14 was completed using the process described in Example 5. The product was obtained as a yellow oil.

Example 18-Distillation of transesterified RBD canola oil Standard procedure for removing more volatile components of fatty acid ethyl ester (FAEE) mixture by vacuum distillation

Fatty acid ethyl ester (FAEE) from RBD canola-DHA (obtained in Example 16) was subjected to distillation under the following conditions. Separation by distillation was accomplished by passing transesterified crude oil through a Pope 2 inch (50 mm) thin film still under vacuum with a 2x1000 ml collection flask for collecting the distillate and residue. The composition of each fatty acid was analyzed.

The vacuum was supplied by the Edwards 3 rotary pump and the vacuum was measured by the ebro pressure gauge VM2000.

The oil was fed to the distiller at 4 mL / min by the Cole-Palmer Instrument Company's easy-load II peristaltic pump, the distiller motor was set to 325 rpm and the distillate was condensed using a water condenser. Supply continued until one of the receiver flasks was full.

The RBD canola-DHA FAEE was distilled under these conditions with the heater band initialized to 152 ° C. to obtain a 50:50 split of distillate: residue. Distillation under standard conditions with the heater band temperature set to 152 ° C. re-used some of the residue from this distillation to remove the more volatile components. The total distillation time was about 90 minutes.

Example 19-Distillation of FAEE from transesterified RBD reference blend Fatty acid ethyl ester (FAEE) from RBD reference blend (obtained in Example 17) under the same conditions as shown in the previous example. Was used for distillation.

The RBD Reference Blend FAEE was distilled under these standard conditions with the heater band initialized to 152 ° C. to obtain a 50:50 split of Distillate: Residue. The residue from this distillation was resubmitted to removal of the more volatile components by distillation under standard conditions. The purpose was to obtain a 50:50 split of Distillate: Residue. Distillation was mainly carried out with the heater band set to 152 ° C. The total distillation time was about 200 minutes.

Chromatographic Separation of FAEE from Example 20-RBD Canola The fatty acid ethyl ester (FAEE) obtained in Example 18 (ie, obtained from RBD canola-DHA and treated using transesterification and distillation). , Was subjected to chromatographic separation under the following conditions. A preparative HPLC system with a Waters Prep 4000 system, a 10 ml loop Rheodyne injector, a 300 x 40 mm Deltaprep C18 column, a Waters2487 dual wavelength detector, and a chart recorder at 70 mL / min in 88% methanol / water mobile phase. Equilibrated. The detector was set to full scale in 215 nm and 2.0 absorbance units and charts were run at 6 cm / hour.

1.0 g of FAEE oil was dissolved in a minimum amount of 88% methanol / water and injected into the column via a Rheodyne injector. When the solvent front appeared after about 7 minutes, about 250 mL fractions were collected.

Analytical HPLC was performed on all fractions, combining "symmetrical" fractions predominantly containing DHA.

Chromatographic Separation of FAEE from Example 21-RBD Reference Blend Distilled fatty acid ethyl ester (FAEE) of RBD reference blend (obtained in Example 19) under the same conditions as shown in the previous example. Was subjected to chromatographic separation.

Analytical HPLC was performed on all fractions, combining "symmetrical" fractions predominantly containing DHA.

Example 22-Fatty Acid Composition Analysis of Concentrated RBD Oil The fatty acid composition of the products obtained in Examples 20 and 21 was analyzed. The results are shown below.

Example 24-Oil Stability Assessment Headspace analysis was performed on the enriched products described in Example 20 and Example 21 according to the method described in Example 12.

The table below summarizes the results obtained from the RBD canola oil obtained in Example 20 and the RBD reference oil obtained in Example 21 at T = 0 to 3 days. The test sample was kept at ambient temperature during this period, on a light box and under fluorescent tube illumination. Analyzing the m / z 58 molecular ions, the mass chromatogram clearly shows the appearance of propanal at 1.37 minutes at room temperature. The table below quantifies the evolution of propanal and the data are shown in Figure 2. DHA canola oil releases substantially a small amount of propanal, demonstrating that the stability of canola oil is improved compared to the reference.

DHA canola oil showed superior stability to oxidation compared to reference oil.

Claims (16)

A vegetable-based lipid composition
(I) Docosahexaenoic acid (22: 6n-3) in an amount of about 50% by weight to about 85% by weight of the total fatty acid content of the composition.
(Ii) With the second polyunsaturated fatty acid in an amount of about 10% by weight to about 90% by weight of the docosahexaenoic acid.
(Iii) Containing a third polyunsaturated fatty acid in an amount of about 10% to about 70% by weight of the second polyunsaturated fatty acid.
The total amount of all other fatty acids in the composition is up to about 20% by weight of the total fatty acid content of the composition, the docosahexaenoic acid, and the second and third polyunsaturated fatty acids, respectively. Are independently provided in the form of fatty acids, fatty acid salts, fatty acid esters, or salts of fatty acid esters, the amount of EPA in the composition being less than 10% by weight of the amount of DHA, a vegetable-based lipid. Composition. The lipid composition according to claim 1, wherein the docosahexaenoic acid is present in an amount of at least about 55% by weight, for example, at least about 60% by weight of the total fatty acid content of the composition. The lipid composition according to claim 1 or 2, wherein the second polyunsaturated fatty acid is present in an amount of about 10% by weight to about 60% by weight of the docosahexaenoic acid. The second polyunsaturated fatty acid is a C18-24 omega-3 polyunsaturated fatty acid containing at least three unsaturated fatty acids, such as α-linolenic acid (18: 3n-3), eicosatetraenoic acid. Claims 1-3, which are polyunsaturated fatty acids selected from the group consisting of (20: 4n-3), docosapentaenoic acid (22: 5n-3), and stearadonic acid (18: 4n-3). The lipid composition according to any one of the above. The lipid according to any one of claims 1 to 4, wherein the third polyunsaturated fatty acid is present in an amount of about 10% by weight to about 65% by weight of the second polyunsaturated fatty acid. Composition. The third polyunsaturated fatty acid is a C18-24 omega-3 polyunsaturated fatty acid containing at least three unsaturated fatty acids, such as α-linolenic acid (18: 3n-3), eicosapentaenoic acid (20). : 5n-3), eicosapentaenoic acid (20: 4n-3), docosapentaenoic acid (22: 5n-3), and stearadonic acid (18: 4n-3). The lipid composition according to any one of claims 1 to 5, which is an unsaturated fatty acid. The DHA and the second and third polyunsaturated fatty acids are provided independently, in the form of fatty acid esters, or salts of fatty acid esters, eg, in the form of fatty acid ethyl esters, or as part of triglycerides. The lipid composition according to any one of claims 1 to 6. The lipid composition according to any one of claims 1 to 7, wherein the lipid composition is derived from a single source. The lipid composition according to any one of claims 1 to 8, wherein the lipid composition is derived from a plant. The plant-based oil is an oil seed, preferably Brassica sp. , Gossypium hersutum, Linum usitatissimum, Helianthus sp. , Cornamus tinctorius, Glycine max, Zea mays, Arabidopsis thaliana, Sorghum bicolor, Sorghum vulgare, Avena sativa, Trifoliaum sp. , Elaesis guineenis, Nicotiana benthamiana, Hordeum vulgare, Lupinus angustifolia, Oryza sativa, Oryza graberrima, Oryza graberrima, Camelina sativa, Camelina, Crambe, Camelina, Crambe. The lipid according to any one of claims 1 to 10, wherein the composition is provided in the form of tablets, capsules, encapsulated gels, ingestible liquids or powders, emulsions, or topical ointments or creams. Composition. The lipid composition according to any one of claims 1 to 11, further comprising one or more additional components selected from the group consisting of antioxidants, stabilizers, and surfactants. A dietary supplement composition comprising the lipid composition according to any one of claims 1 to 12. A food product for human or animal consumption, which comprises the lipid composition according to any one of claims 1 to 13, wherein the DHA and the second and third polyunsaturated fatty acids are triglycerides. Foods provided in the form of esters. Used to treat or prevent cardiovascular disease, protect against death in patients with cardiovascular disease, lower overall serum cholesterol levels, lower hypertension, increase HDL: LDL ratios, lower triglycerides, or lower apolipoprotein B levels The vegetable-based lipid composition according to any one of claims 1 to 12. The process for producing the vegetable-based lipid composition according to any one of claims 1 to 12, providing a mixture of fatty acid ethyl esters and subjecting the mixture to a chromatography separation process. And, optionally, a mixture of said fatty acid ethyl esters obtained by ester exchange and distillation of a vegetable-based lipid oil.

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